RESUMEN
Translocation of intrauterine contraceptive device (IUD) from the uterus rarely occurs, which can lead to serious complications. Here the authors reported three cases of IUD migration from into the ureter, bladder, and peritoneal cavity that caused hydronephrosis, respectively. All the three patients received minimally invasive surgeries and recovered.
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Hidronefrosis , Migración de Dispositivo Intrauterino/efectos adversos , Dispositivos Intrauterinos/efectos adversos , Procedimientos Quirúrgicos Operativos/métodos , Adulto , Femenino , Humanos , Hidronefrosis/diagnóstico , Hidronefrosis/etiología , Hidronefrosis/cirugía , Cavidad Peritoneal/diagnóstico por imagen , Cavidad Peritoneal/patología , Cavidad Peritoneal/cirugía , Resultado del Tratamiento , Uréter/diagnóstico por imagen , Uréter/patología , Uréter/cirugía , Vejiga Urinaria/diagnóstico por imagen , Vejiga Urinaria/patología , Vejiga Urinaria/cirugíaRESUMEN
An alternating integrator has been designed for the Experimental Advanced Superconducting Tokamak that is intended for long pulse operation of up to 1000 s. The electromagnetic operating environment for the device is so complex that it could affect the performance of the integrator. The new integrator system is carefully designed and actualized based on specific reduced electromagnetic interference requirements, which were formulated based on consideration of processing of the input signals, the isolation properties, and the circuit board layout and grounding. The developed integrator shows excellent electromagnetic compatibility and low-drift properties.
RESUMEN
The compilation of medical bamboo slips, Ci shu(Needling Methods), which was unearthed from a Han tomb in Mt. Laoguan, is a monograph dealing exclusively with the principles of clinical acupuncture manipulations with 40 acupuncture prescriptions, being the earliest unearthed work with documented standard methods of acupuncture manipulations and acupuncture prescriptions in China. The chapter Zhen fang (Acupuncture Prescriptions) is the earliest summary of standardized acupoint prescriptions up to now in China, which is of great significance to clinical practice directly derived from ancient clinical performance of acupuncture. The chapter Zhen fang of the book Ci shu is also one of the earliest ancient clinical reports archiving the acupoint. This may provide invaluable perspectives to the study of the conceptualization, origination, development, formation of theoretical system, and clinical application of acupoints.
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Acupuntura/historia , Manuscritos como Asunto , Puntos de Acupuntura , Terapia por Acupuntura , Entierro , China , Historia AntiguaRESUMEN
In general, the phospholipase C (PLC) signaling pathway is involved in many physiological activities, including cell growth. However, little is known regarding how the PLC signaling pathway participates in regulating hepatocyte (HC) growth during liver regeneration (LR). To further explore the influence of the PLC signaling pathway on HCs at the cellular level, HCs of high purity and vitality were isolated using Percoll density-gradient centrifugation after partial hepatectomy. The genes of the PLC signaling pathway and target genes of transcription factors in the pathway were obtained by searching the pathways and transcription factor databases, and changes in gene expression of isolated HCs were examined using the Rat Genome 230 2.0 Microarray. The results suggested that various genes involved in the pathway (including 151 known genes and 39 homologous genes) and cell growth (including 262 known genes and 37 homologous genes) were associated with LR. Subsequently, the synergetic effect of these genes in LR was analyzed using a mathematical model (Et) according to their expression profiles. The results showed that the Et values of G protein-coupled receptor/PLC, integrin/PLC, and growth factor receptor/PLC branches of the PLC pathway were all significantly strengthened during the progression and termination phases of LR. The synergetic effect of target genes, in parallel with target gene-related cell growth, was also enhanced during whole rat LR, suggesting the potential positive effect of PLC on HC growth. The present data indicate that the PLC signaling pathway may promote HC growth through 3 mechanisms during rat LR after partial hepatectomy.
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Regeneración Hepática/genética , Transducción de Señal/genética , Fosfolipasas de Tipo C/genética , Animales , Proliferación Celular/genética , Hepatocitos/metabolismo , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Factores de Transcripción/genética , Fosfolipasas de Tipo C/aislamiento & purificación , Fosfolipasas de Tipo C/metabolismoRESUMEN
A high-performance integrator is one of the key electronic devices for reliably controlling plasma in the experimental advanced superconducting tokamak for long pulse operation. We once designed an integrator system of real-time drift compensation, which has a low integration drift. However, it is not feasible for really continuous operations due to capacitive leakage error and nonlinearity error. To solve the above-mentioned problems, this paper presents a new alternating integrator. In the new integrator, the integrator system of real-time drift compensation is adopted as one integral cell while two such integral cells work alternately. To achieve the alternate function, a Field Programmable Gate Array built in the digitizer is utilized. The performance test shows that the developed integrator with the integration time constant of 20 ms has a low integration drift (<15 mV) for 1000 s.
RESUMEN
N-terminal acetylation is the most common modification in eukaryotic proteins, affecting stability and activity of proteins. NatA is one of the N-terminal acetytransferases in yeast. It is composed of two subunits, NAT1 and ARD1. Defect in one of them leads to loss of activity of NatA. Null mutant of NAT1 in yeast exhibits a variety of phenotypes, including depression of a silent mating type locus (HML), failing to enter G(0) in poor nutrient situations and chromosomes instability. Based on homology of NAT1 between yeast and other organisms, the full-length CDS (coding sequence) of HNAT1 was cloned and sequenced. Result of in situ hybridization in testis of rat showed that expression of NAT1 was high and its expression was different in different phases of spermatogenesis. The gene may play an important role in spermatogenesis.
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Acetiltransferasas/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Espermatogénesis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Especificidad de la EspecieRESUMEN
PURPOSE: To determine the effect of interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha on cytokine, chemokine, and receptor expression in corneal stromal cells; the effect of corneal scrape injury on monocyte chemotactic and activating factor (MCAF) expression and monocyte-macrophage influx into the stroma; and the effect of MCAF and granulocyte colony-stimulating factor (G-CSF) microinjection on inflammatory cell infiltration into the stroma. METHODS: Gene array technology was used to evaluate changes in cytokine, chemokine, and receptor gene expression in stromal fibroblasts in response to IL-1alpha and TNFalpha. Expression of MCAF mRNA and protein was monitored with an RNase protection assay and Western blot analysis, respectively. Keratocyte MCAF protein expression in the rabbit cornea was detected with immunocytochemistry. After epithelial scrape injury, monocytes-macrophages were detected in rabbit corneas, by immunocytochemistry for monocyte-macrophage antigen. Inflammatory cell infiltration after MCAF and G-CSF microinjection into the stroma of mouse corneas was monitored with hematoxylin and eosin staining. RESULTS: IL-1alpha or TNFalpha upregulated the expression of several proinflammatory chemokines in stromal fibroblasts in culture. These included G-CSF, MCAF, neutrophil-activating peptide (ENA-78), and monocyte-derived neutrophil chemotactic factor (MDNCF). MCAF mRNA upregulation was confirmed by RNase protection assay, and MCAF protein was detected by Western blot analysis. MCAF protein was detected in keratocytes at 4 hours and 24 hours after epithelial injury, but not in keratocytes in the unwounded cornea. Corneal epithelial injury triggered the influx of monocytes-macrophages into the corneal stroma in the rabbit. Microinjection of MCAF and G-CSF into mouse cornea resulted in the influx of monocytes-macrophages and granulocytes, respectively, into the stroma. CONCLUSIONS: Proinflammatory chemokine induction in keratocytes is mediated by IL-1alpha and TNFalpha. The proinflammatory chemokines produced by the keratocytes probably trigger the influx of inflammatory cells into the stroma after epithelial injury associated with corneal surgery, contact lenses, or trauma.
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Movimiento Celular/fisiología , Quimiocinas/biosíntesis , Sustancia Propia/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Interleucina-1/farmacología , Macrófagos/fisiología , Monocitos/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Western Blotting , Quimiocina CCL2/farmacología , Quimiocinas/genética , Sustancia Propia/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos/farmacología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , Receptores CCR2 , Receptores de Quimiocina/biosíntesis , Regulación hacia ArribaRESUMEN
In the vertebrate retina, the final step of visual chromophore production is the oxidation of 11-cis-retinol to 11-cis-retinal. This reaction is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs), prior to the chromophore rejoining with the visual pigment apo-proteins. The RDH5 gene encodes a dehydrogenase that is responsible for the majority of RDH activity. In humans, mutations in this gene are associated with fundus albipunctatus, a disease expressed by delayed dark adaptation of both cones and rods. In this report, an animal model for this disease, 11-cis-rdh-/- mice, was used to investigate the flow of retinoids after a bleach, and microsomal membranes from the retinal pigment epithelium of these mice were employed to characterize remaining enzymatic activities oxidizing 11-cis-retinol. Lack of 11-cis-RDH leads to an accumulation of cis-retinoids, particularly 13-cis-isomers. The analysis of 11-cis-rdh-/- mice showed that the RDH(s) responsible for the production of 11-cis-retinal displays NADP-dependent specificity toward 9-cis- and 11-cis-retinal but not 13-cis-retinal. The lack of 13-cis-RDH activity could be a reason why 13-cis-isomers accumulate in the retinal pigment epithelium of 11-cis-rdh-/- mice. Furthermore, our results provide detailed characterization of a mouse model for the human disease fundus albipunctatus and emphasize the importance of 11-cis-RDH in keeping the balance between different components of the retinoid cycle.
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Oxidorreductasas de Alcohol/metabolismo , Epitelio Pigmentado Ocular/enzimología , Vitamina A/metabolismo , Oxidorreductasas de Alcohol/deficiencia , Oxidorreductasas de Alcohol/genética , Animales , Quimera , Cruzamientos Genéticos , Oscuridad , Femenino , Genotipo , Membranas Intracelulares/metabolismo , Cinética , Luz , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Microsomas/metabolismo , Oxidación-Reducción , Ácido Palmítico/metabolismo , Retinoides/aislamiento & purificación , Retinoides/metabolismo , Especificidad por SustratoRESUMEN
Mutations in the retinal pigment epithelium gene encoding RPE65 are a cause of the incurable early-onset recessive human retinal degenerations known as Leber congenital amaurosis. Rpe65-deficient mice, a model of Leber congenital amaurosis, have no rod photopigment and severely impaired rod physiology. We analyzed retinoid flow in this model and then intervened by using oral 9-cis-retinal, attempting to bypass the biochemical block caused by the genetic abnormality. Within 48 h, there was formation of rod photopigment and dramatic improvement in rod physiology, thus demonstrating that mechanism-based pharmacological intervention has the potential to restore vision in otherwise incurable genetic retinal degenerations.
Asunto(s)
Ceguera/tratamiento farmacológico , Epitelio Pigmentado Ocular/fisiopatología , Proteínas/fisiología , Degeneración Retiniana/tratamiento farmacológico , Retinaldehído/uso terapéutico , Administración Oral , Animales , Ceguera/fisiopatología , Proteínas Portadoras , Niño , Modelos Animales de Enfermedad , Diterpenos , Proteínas del Ojo , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/fisiopatología , Retinaldehído/administración & dosificación , Retinaldehído/metabolismo , Retinoides/administración & dosificación , Retinoides/metabolismo , Retinoides/uso terapéutico , Factores de Tiempo , cis-trans-IsomerasasRESUMEN
Disseminated superficial actinic porokeratosis is an autosomal dominant cutaneous disorder characterized by many uniformly small, minimal, annular, anhidrotic, and keratotic lesions. The genetic basis for this disease is unknown. Using a genomewide search in a large Chinese family, we identified a locus at chromosome 12q23.2-24. 1 responsible for disseminated superficial actinic porokeratosis. The fine mapping study indicates that the disseminated superficial actinic porokeratosis gene is located within a 9.6 cM region between markers D12S1727 and D12S1605, with a maximum two-point LOD score of 20.53 (theta = 0.00) at D12S78. This is the first locus identified for a genetic disease where the major phenotype is porokeratosis. The study provides a map location for isolation of a gene causing disseminated superficial actinic porokeratosis.
Asunto(s)
Poroqueratosis/genética , China , Mapeo Cromosómico , Cromosomas Humanos Par 12 , Humanos , Escala de Lod , Repeticiones de Microsatélite/genética , Linaje , Recombinación GenéticaRESUMEN
PURPOSE: Tissue-cultured corneal epithelial transplantation is a novel procedure that uses tissue-cultured epithelial cells to restore severely damaged ocular surfaces. In this study, we used tissue-cultured human limbal and amniotic epithelial cells as donor cells to investigate the feasibility of this procedure for reestablishment of a damaged ocular surface in experimental conditions. METHODS: Primary human limbal epithelial cultures were established from banked limbal tissue. Amniotic epithelial cells were isolated from serologically screened human placenta and maintained in a specialized nutrient medium. Suspended cells (5 x 10(5)/ml) were seeded onto the concave surface of collagen corneal shields and incubated at 37 degrees C for 2-3 days. These cell-covered shields were then placed on a denuded stromal surface in organ culture and on New Zealand albino rabbit ocular surfaces that had the native epithelium previously removed. Specimens were collected 24, 48, 72, and 96 h later from organ-cultured corneal buttons and recipient animals, processed, and evaluated histologically. RESULTS: The cells grown on the collagen shield were spread uniformly and unpolarized after 48 h in culture. They were repolarized and tightly adhered to the recipient corneal stroma 24 h after transplantation, as demonstrated by formation of cell-substrate hemidesmosomes (HDs) and donor-specific antigen immunostaining. The donor cells were retained in six of 15 rabbits receiving limbal cells and four of 12 rabbits receiving amniotic cells for as long as 10 days after surgery. CONCLUSION: Cultured human limbal and amniotic epithelial cells can be successfully transplanted onto a denuded corneal surface where they adhere tightly to underlying stroma by hemidesmosomes.
Asunto(s)
Amnios/citología , Proteínas Portadoras , Trasplante de Células/métodos , Colágeno , Enfermedades de la Córnea/cirugía , Sustancia Propia/cirugía , Proteínas del Citoesqueleto , Células Epiteliales/trasplante , Limbo de la Córnea/citología , Proteínas del Tejido Nervioso , Colágenos no Fibrilares , Animales , Autoantígenos/análisis , Adhesión Celular , Células Cultivadas , Enfermedades de la Córnea/patología , Sustancia Propia/ultraestructura , Medios de Cultivo Condicionados , Desmosomas/ultraestructura , Distonina , Células Epiteliales/química , Técnica del Anticuerpo Fluorescente Indirecta , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Técnicas de Cultivo de Órganos , Conejos , Colágeno Tipo XVIIRESUMEN
We sought to develop corneal endothelial cell cultures with extended lifespan from corneas with Fuchs' dystrophy. Descemet's-endothelial cell explants from histology confirmed recipient corneas of two patients with Fuchs' dystrophy were cultured. After a small number of corneal endothelial cells with irregular, endothelial morphology migrated from the explants onto the culture plate, the cells were transduced with a disabled retrovirus (pLXSN16E6/E7) coding for the human papilloma virus type 16 transforming oncoproteins E6 and E7. Expression of E6/E7 mRNA in the cell cultures with extended lifespan was monitored by RT-PCR. In vitro labeled cellular protein patterns of the Fuchs' E6/E7 cell cultures with extended lifespan were compared with those from normal human corneal endothelial E6/E7 cell cultures with extended lifespan using two-dimensional gel electrophoresis. Two endothelial cell cultures with extended lifespan were derived from Fuchs' corneas. The morphology of the Fuchs' cells expressing E6 and E7 was similar to that of normal corneal endothelial cells expressing E6 and E7. Surprisingly, the rate of proliferation of Fuchs'-derived cells was similar to that of normal endothelial cells transduced with E6/E7. Proliferation of each Fuchs' cell culture with extended lifespan continued for over 30 population doublings. There were limited quantitative differences in the two-dimensional gel electrophoretic protein patterns of the Fuchs'-derived and normal endothelial cell cultures with extended lifespan, respectively. Retroviral integration is dependent on cell proliferation. Thus, cells that migrated from the Fuchs' Descemet's explants were undergoing at least limited in vitro proliferation when the retroviral vector coding for E6/E7 integrated. Fuchs' corneal endothelial cells expressing E6 and E7 had similar proliferation, cellular morphology, and two-dimensional gel protein electrophoretic patterns to normal corneal endothelial cells expressing the same oncoproteins.
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Distrofia Endotelial de Fuchs/patología , División Celular , Línea Celular Transformada/metabolismo , Endotelio Corneal/ultraestructura , Distrofia Endotelial de Fuchs/metabolismo , Humanos , Microscopía Electrónica , Proteínas Oncogénicas/metabolismo , Oncogenes , Papillomaviridae/genética , Transcripción GenéticaRESUMEN
The purpose of this study was to determine the role of programmed cell death (apoptosis) in the disappearance of keratocytes beneath an epithelial debridement wound in the cornea and to investigate a potential role of interleukin-1 (IL-1) in induction of apoptosis in stromal fibroblasts in vitro and keratocytes in vivo. Keratocyte and stromal fibroblast cell morphology was examined in wounded and unwounded mouse corneas using transmission electron microscopy. Nuclear DNA fragmentation was detected with the TUNEL assay for 3'-hydroxyl DNA ends. The effect of IL-1 on keratocytes in vivo was determined by microinjection of IL-1 alpha into the central corneal stroma via a limbal entry site. The in vitro effects of interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) were determined with primary cultures of human corneal stromal and dermal fibroblasts. Cell shrinkage, blebbing with formation of membrane bound bodies, condensation and fragmentation of the chromatin, and DNA fragmentation, consistent with apoptosis were detected in anterior stromal keratocytes after epithelial scrape wounds. Thus, disappearance of keratocytes from the underlying stroma following epithelial debridement is mediated by apoptosis. Microinjection of IL-1 alpha into the central stroma of the mouse cornea caused a redistribution of keratocytes in the stroma via apoptosis and, possibly, negative chemotaxis. IL-1 alpha and IL-1 beta induced apoptosis in corneal stromal and dermal fibroblasts in vitro. The epithelial/endothelial-stromal IL-1 system may mediate corneal tissue organization and responses to mechanical- and pathogen-induced injury through induction of keratocyte apoptosis. Keratocyte apoptosis is likely an initiating event in wound healing following corneal surgery. We hypothesize that derangement's in this system may have a role in the pathogenesis of keratoconus and other diseases of the cornea.
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Apoptosis , Lesiones de la Cornea , Interleucina-1/farmacología , Cicatrización de Heridas/fisiología , Animales , Apoptosis/efectos de los fármacos , Recuento de Células , Células Cultivadas , Córnea/efectos de los fármacos , Córnea/ultraestructura , ADN/análisis , Epitelio/lesiones , Fibroblastos/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Factores de TiempoRESUMEN
The present study examined the potential of orally induced tolerance for preventing immunological rejection of corneal allografts. Orthotopic corneal allografts were transplanted from either C3H (MHC + multiple minor H-mismatched) or NZB (multiple minor H-mismatched only) donors to CB6F1 recipients on day 0. Tissue cultured corneal epithelial and endothelial cells from relevant donor strains were administered orally from day -14 to day -4 on a daily basis, The incidence of graft rejection, graft mean survival time (MST), and alloimmune responses, and the antigen specificity of induced tolerance were studied. Oral immunization induced a remarkable tolerance such that only 55% of the orally immunized hosts rejected their fully allogeneic corneal grafts (MST = 43 days) compared with 100% rejection (MST = 18 days) in normal controls. Likewise, rejection of MHC-matched, multiple minor H-mismatched corneal grafts fell from 80% in untreated controls to 36% in orally immunized hosts. Oral immunization was effective in desensitizing previously immunized hosts. Rejection of MHC-matched, multiple H minor-mismatched corneal allografts fell from 93% in preimmune, unfed hosts to 36% in preimmune, orally tolerized mice. Thus, oral immunization is a safe and effective method for desensitizing high-risk, preimmune hosts and promoting corneal allograft survival.
Asunto(s)
Córnea/inmunología , Trasplante de Córnea/inmunología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/inmunología , Inmunización , Terapia de Inmunosupresión/métodos , Administración Oral , Animales , Córnea/citología , Epitelio/inmunología , Epitelio/trasplante , Epítopos , Femenino , Rechazo de Injerto/inmunología , Hipersensibilidad Tardía/inmunología , Incidencia , Isoanticuerpos/biosíntesis , Isoantígenos/uso terapéutico , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos NZB , Linfocitos T Citotóxicos/inmunología , Factores de Tiempo , Trasplante Homólogo/inmunologíaRESUMEN
A mouse model of penetrating keratoplasty was used to evaluate the efficacy of ultraviolet radiation (UVR) and hyperbaric oxygen (HBO) treatments in depleting corneal Langerhans cells (LC) and promoting corneal allograft survival. The presence of donor-derived LC dramatically increased the immunogenicity and rejection rate of corneal allografts. Rejection increased from 40% in LC- corneal grafts to 80% in grafts containing donor-derived LC. Pretreatment with either HBO or UVR resulted in a sharp decrease in both the incidence and tempo of rejection for grafts containing donor LC, but neither procedure affected the fate of LC- corneal allografts. UVR-treatment abolished the immunogenicity of LC+ grafts. UVR-treated orthotopic grafts failed to elicit either cytotoxic T lymphocyte (CTL) or delayed-type hypersensitivity (DTH) responses that were any greater than naive control mice. The results suggest that purging corneal allografts of stray donor-derived LC might improve corneal allograft survival in high-risk patients without jeopardizing the functional integrity of the graft.
Asunto(s)
Trasplante de Córnea , Supervivencia de Injerto , Células de Langerhans/inmunología , Donantes de Tejidos , Formación de Anticuerpos , Córnea/citología , Córnea/inmunología , Rechazo de Injerto/epidemiología , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/efectos de la radiación , Humanos , Oxigenoterapia Hiperbárica , Incidencia , Isoanticuerpos/inmunología , Células de Langerhans/efectos de los fármacos , Células de Langerhans/efectos de la radiación , Linfocitos T/inmunología , Trasplante Homólogo , Rayos UltravioletaRESUMEN
PURPOSE: Human corneal endothelial cells are thought to have limited capacity for proliferation. Little is known about the mechanisms that regulate the proliferation of these cells. The authors introduced oncogenes into human corneal endothelial cells to modulate proliferation. In addition, they sought to establish cell lines to facilitate study of human corneal endothelial cells. METHODS: Early-passage human corneal endothelial cells were transduced with disabled retrovirus (pLXSN16E6/E7) coding for the human papilloma virus type 16 transforming oncoproteins E6 and E7. Early-passage cells were also stably transfected by electroporation with the pMTV-D305 plasmid vector, in which SV40 large T antigen (SV40 LTAg) mRNA expression is positively regulated by the mouse mammary tumor virus promoter. Expression of E6/E7 mRNA or SV40 LTAg mRNA in cell lines was monitored with the polymerase chain reaction. SV40 LTAg protein expression was detected by immunocytology and Western blot analysis. RESULTS: Human corneal endothelial cells were efficiently infected with disabled retrovirus coding for E6/E7, and seven strains of cells have continued active proliferation for more than 50 population doublings (PD) (< 8 control PD). E6/E7 mRNA was expressed by each cell strain. E6/E7 transformed cells proliferate rapidly and form a monolayer of cells with a high degree of contact inhibition. Transfection with pMTV-D305 is less efficient, and only a single strain was developed. pMTV-D305-transfected endothelial cells (dexamethasone induced) proliferated at a lower rate than E6/E7-transduced cells or cells transfected with a vector (pSV3neo) in which SV40 LTAg is constitutively regulated. In the absence of dexamethasone, the proliferation of pMTV-D305-transfected cells was even slower, but cells continued to produce SV40 LTAg mRNA and protein. The latter results indicated that SV40 LTAg mRNA continued to be synthesized at significant levels in pMTV-D305-transfected cells in the absence of the inducer dexamethasone. CONCLUSIONS: This study suggests that human corneal endothelial cells have a high capacity for proliferation. Thus, cell division is normally controlled in human corneal endothelial cells by poorly characterized, but efficient, mechanisms. Because the E6 and E7 proteins, as well as the SV40 large T antigen, specifically bind to and interfere with the activity of the retinoblastoma (RB) and p53 tumor suppressor proteins, our results suggest that these proteins have critical roles in regulating the proliferation of human corneal endothelial cells.
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Antígenos Transformadores de Poliomavirus/biosíntesis , Endotelio Corneal/citología , Endotelio Corneal/metabolismo , Proteínas Oncogénicas Virales/biosíntesis , Oncogenes , Proteínas Represoras , Antígenos Transformadores de Poliomavirus/genética , Secuencia de Bases , División Celular , Línea Celular , Transformación Celular Viral , Preescolar , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , ARN Viral/análisis , TransfecciónRESUMEN
We sought to determine the effects of exogenous epidermal growth factor (EGF), heparin-binding EGF (HB-EGF), transforming growth factor alpha (TGF-alpha), single-chain precursor hepatocyte growth factor (SC-HGF), double-chain mature HGF (DC-HGF), and keratinocyte growth factor (KGF) on proliferation, motility, and differentiation of first passage cultures of human corneal epithelial cells in serum-free chemically defined medium. The effect of EGF, HB-EGF, TGF-alpha, SC-HGF, DC-HGF, KGF or combinations of the growth factors on proliferation was measured by counting cells present after 3 weeks of culture and by immunostaining for the cell-cycle-specific nuclear proliferation antigen Ki-67. The effect of the factors on epithelial cell motility was assessed by morphometric analysis of photographs of cells migrating from confluent islands of cells. The effect of growth factors on differentiation of epithelial cells were determined by immunostaining epithelial cell islands for the keratin K3 and by Western blotting for keratin K3. EGF, alone or in combination with KGF and SC-HGF, significantly stimulated motility of epithelial cells at the periphery of confluent islands of cells and induced an elongated cell morphology. TGF-alpha, HB-EGF and DC-HGF produced motility effects similar to EGF. There was diminished proliferation of the migrating cells in response to EGF, HB-EGF, TGF-alpha or DC-HGF, while non-migrating epithelial cells in the center of confluent islands continued to proliferate in response to the growth factors. EGF, HB-EGF, TGF alpha or DC-HGF inhibited expression of the differentiation-related marker keratin K3 in epithelial cells, both at the edge and at the center of the islands. KGF stimulated proliferation of corneal epithelial cells at low density and in confluent islands of cells. KGF did not affect expression of keratin K3 or migration of epithelial cells. SC-HGF had no effect on corneal epithelial cells. These results indicate that the effects of EGF, HB-EGF, TGF-alpha and DC-HGF on corneal epithelial cell proliferation, motility and differentiation vary from those of KGF and SC-HGF. EGF, HB-EGF, TGF-alpha and DC-HGF induced changes in epithelial cell morphology and motility in cells plated at low cell density or in cells located at the edge of a confluent island. Thus, these effects appear to be dependent on the extent of cell-cell contact. The inhibitory effect of EGF, HB-EGF, TGF-alpha or DC-HGF on corneal epithelial cell differentiation, however, is independent of cell density.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Córnea/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Western Blotting , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Epitelio/efectos de los fármacos , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Humanos , Mitosis/efectos de los fármacosRESUMEN
The purpose of this study was to determine whether epidermal growth factor (EGF), EGF receptor, transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-beta), acidic fibroblast growth factor (acidic-FGF), basic fibroblast growth factor (basic-FGF), and interleukin-1-alpha (IL-1-alpha) proteins were present in cultures of human corneal cells and/or in sections of human corneal tissue. Immunohistochemistry was performed on human corneal sections. Immunofluorescent cell staining was used to evaluate corneal epithelial, stromal fibroblast, and endothelial cells in primary culture. Basic-FGF production was evaluated in culture cells using immunoprecipitation. EGF, TGF-alpha, TGF-beta-1, and IL-1-alpha were detected by immunohistochemistry in cells in all three layers of the cornea. EGF receptor and acidic FGF were detected by immunohistochemistry in epithelial and endothelial cells, but not in stromal fibroblast cells. Differences in distribution of the growth factors were noted within individual layers of the cornea. EGF and basic-FGF proteins were detected in all three predominant cell types of the cornea using immunocytology. IL-1-alpha protein was detected by immunocytology in corneal epithelial and endothelial cells, but not stromal fibroblasts. Immunoprecipitation confirmed the production of basic-FGF in all three cell types. IL-1-alpha protein detection in the corneal stroma by immunohistology, but not by immunocytology in first passage stromal fibroblasts, suggests that IL-1-alpha may localize to the corneal stroma after production by corneal epithelial and/or endothelial cells.
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Córnea/química , Sustancias de Crecimiento/análisis , Adulto , Células Cultivadas , Endotelio Corneal/química , Factor de Crecimiento Epidérmico/análisis , Factor 1 de Crecimiento de Fibroblastos/análisis , Factor 2 de Crecimiento de Fibroblastos/análisis , Humanos , Inmunohistoquímica , Interleucina-1/análisis , Pruebas de Precipitina , Factor de Crecimiento Transformador alfa/análisis , Factor de Crecimiento Transformador beta/análisisRESUMEN
Interleukin-1 receptor and glucocorticoid receptor messenger ribonucleic acid (RNA) sequences coding for the corresponding proteins were detected in corneal epithelium, stromal fibroblast, and endothelial cells using the polymerase chain reaction and hot blotting. Identification of interleukin-1 receptor mRNA in each of the three major cell types of the cornea suggests that interleukin-1 alpha has autocrine and/or paracrine roles in the cornea, since previous studies have found that interleukin-1 alpha mRNA is produced in corneal epithelial, stromal fibroblast, and endothelial cells. Further investigation is needed to determine the functions regulated by the interleukin-1 receptor and glucocorticoid receptor in the cornea and the role of each in corneal wound healing.
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Córnea/metabolismo , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Interleucina-1/metabolismo , Adolescente , Adulto , Secuencia de Bases , División Celular , Células Cultivadas , Niño , Preescolar , Córnea/citología , Sustancia Propia/metabolismo , Cartilla de ADN , Endotelio Corneal/metabolismo , Epitelio/metabolismo , Fibroblastos/metabolismo , Humanos , Immunoblotting , Lactante , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: The purpose of this article is to introduce the practicing ophthalmologist to the optical principles and images produced by a tandem scanning confocal microscope (recently approved by the Food and Drug Administration for general clinical use). The tandem scanning confocal microscope allows real-time viewing of structures in the living cornea at the cellular level in four dimensions (x, y, z, and time). METHODS: Nine patients (2 males, 7 females), ranging in age from 7 to 52 years, were examined. Images were recorded on super VHS videotape, digitized and processed on a computer workstation, and photographed for presentation. RESULTS: Two-dimensional (x, y) 400 x 400-microns images (9-microns z-axis thickness) are presented for normal corneal structures and for the clinical conditions of herpetic keratitis, wound healing after myopic excimer ablation, Acanthamoeba infection, corneal dystrophies (granular, Reis-Buckler), contact lens abrasion, and the irido-corneal endothelial syndrome. CONCLUSION: Clinical confocal microscopy has the unique potential of providing noninvasive assessment of corneal injury and disease at the cellular level that is not available currently from other technologies.